Pharmaceutical preparations of a variety of physiologically active peptides or physiologically proteins are currently available in the market place. Among such physiologically active peptides or physiologically proteins are insulin, growth hormones, atrial natriuretic peptide, calcitonin, LHRH analogues, parathyroid hormone, and adrenocorticotropic hormone derivatives. Since these compounds are deactivated in the gastrointestinal tract by the action of proteases, they are rarely administered in oral preparations; most are formulated as parenteral preparations such as injections for clinical use.
Continuous, long-term treatment of diseases by injection requires patients to visit a hospital or clinic to receive treatment and poses a significant burden to patients. Thus, subcutaneous injections that patients can administer themselves have been desired. Drugs administered by subcutaneous injection, however, are decomposed by proteases during the subcutaneous absorption process and their bioavailability generally becomes lower than is achieved by intravenous injection.
To compensate for the low bioavailability of drugs, currently used subcutaneous injections contain increased doses of drugs or require frequent administration as compared to the intravenous administration. Thus, subcutaneous injections still remain stressful to patients. As used herein, the term “biological availability (EA)” refers to how much of the administered drug reaches the blood circulation (also referred to as extent of bioavailability (EBA)).
Ghrelin, an endogenous growth hormone secretagogue (GHS) that binds to growth hormone secretagogue receptor (GHS-R), is a physiologically active peptide first isolated from rat stomach in 1999 (Non-Patent Document 1). Ghrelins with similar primary structures were later isolated from, or suggested by cDNA analysis to be present in, other vertebrates, including human, mice, pigs, chickens, eel, cows, horses, sheep, frogs, rainbow trout and dogs. Their primary structures were shown in Table 1. Ghrelin has also been isolated from cats and goats.
TABLE 1HumanGSS(n-octanoyl)FLSPEHQRVQQRKESKKPPAEIQPRGSS(n-octanayl)FLSPEHQRWREESKKPPAKLQPR RatGSS(n-octanoyl)FLSPEHQKAQQRKESEKPPAKLQPRGSS(n-octanoyl)FLSPEHQKAQREESKKPPAKLQPR MouseGSS(n-octanoyl)FLSPEHQKAQQREESKKPPAKLQPR PorcineGSS(n-octanoyl)FLSPEHQKVQQRKESEXPAAKLKPR BovineGSS(n-octanoyl)FLSPEHQKLQRKEAKKPSGRLKPR OvineGSS(n-octanoyl)FLSPEHQKLQREEPKKPSGRLKPR CanineGSS(n-octanoyl)FLSPEHQKLQQREESKKPPAKLQPR EelGSS(n-octanoyl)FLSPSQRPQGRDEYPPRV-NH2 TroutGSS(n-octanoyl)FLSPSQKPQVRQGKGKPPRV-NH2GSS(n-octanoyl)FLSPSQKPQGKGKPPRV-NH2 ChickenGSS(n-octanoyl)FLSPTYKNIQQQEGTRKPTARGSS(n-octanoyl)FLSPTYKNIQQQEDTRKPTARGSS(n-octanoyl)FLSPTYKNIQQQEDTRKPTARLH BullfrogGLT(n-octanoyl)FLSPADMQKIAERQSQNKLRHGNMGLT(n-decanoyl)FLSPADMQKIAERQSQNKLRHGNMGLT(n-octanoyl)FLSPADMQKIAERQSQNKLRHGNMN TilapiaGSS(n-octanoyl)FLSPSQKPQNKVKSSRI-NH2 CatfishGSS(n-octanoyl)FLSPTQKPQNRGDRKPPRV-NH2GSS(n-octanoyl)FLSPTQKPQNRGDRKPPRVG EquineGSS(n-butanoyl)FLSPEHHKVQHREESKKPPAKLKPR(In Table 1, amino acid residues are indicated by IUPAC/TUC single letter codes.)
Endogenous ghrelin found in these animals is a peptide that has a unique hydrophobic modified structure in which the serine (S) or threonine (T) residue at position 3 is acylated with a fatty acid such as octanoic acid and decanoic acid. Ghrelin binds to growth hormone secretagogue receptor to increase the intracellular level of calcium ions. Studies have revealed that ghrelin is a potent growth hormone secretagogue that modulates the secretion of growth hormone. Thus, the physiological roles and potential pharmaceutical applications of ghrelin have attracted significant interest (Patent Document 1). In the present invention, all types of naturally occurring ghrelins are collectively referred as “ghrelin.”
Ghrelin derivatives or analogues obtained by partial deletion or substitution of natural animal ghrelin have also drawn much attention as treatment for various diseases (Patent Document 3).
In ghrelin derivatives, the hydrophobic modified structure does not contain an octanoyl group (C8) as in natural ghrelin, but a different modified structure, such as that containing a fatty acid having 2 to 20, preferably about 4 to 12 carbon atoms, including, for example, a hexanoyl group (C6), a decanoyl group (C10) and a dodecanoyl group (C12), that containing a fatty acid branched or unsaturated derivatives thereof, that containing an aromatic ring, such as a phenylpropionyl group, and that containing an adamantane backbone. While the hydrophobic modified structure of natural animal ghrelin is bound to the peptide backbone via an ester linkage, this linkage may be provided by an ester, ether, thioether, amide, or disulfide linkage in ghrelin derivatives (Patent Document 1).
Despite high expectations for application of ghrelins comprising ghrelin, ghrelin derivatives and ghrelin analogues in pharmaceutical products, no viable pharmaceutical compositions have yet to be designed and much still remains unknown about the pharmacokinetics of the compounds.
As to the pharmaceutical compositions using ghrelins, Patent Document 2 describes the effect of pH on aqueous solutions of ghrelins, as well as a method for preventing decomposition of the hydrophobic modified structure of ghrelins. According to Patent Document 2, ghrelins are stable in an aqueous solution in a pH range of 2 to 7. The pH of the solution can be adjusted by pH adjusters and buffers. The buffer is used to minimize the pH change of the aqueous solution during storage. Among the buffers that are described in Patent Document 2 are glycine-hydrochloric acid buffer, acetic acid buffer, citric acid buffer, lactic acid buffer, phosphoric acid buffer, citric acid-phosphoric acid buffer, phosphoric acid-acetic acid-boric acid buffer and phthalic acid buffer (Patent Document 2, p. 11, lines 5-14).
In examples described in Patent Document 2, the stability of ghrelin is tested in McIlvaine buffer (a mixture of aqueous citric acid and an aqueous solution of disodium hydrogen phosphate), Britton-Robinson buffer (a mixture of aqueous phosphoric acid/acetic acid/boric acid and an aqueous sodium hydroxide solution), citric acid buffer, glycine-hydrochloric acid buffer and acetic acid buffer. While Patent Document 2 describes that the stability of ghrelins in each solution was ensured by maintaining the pH of the solution within a range of 2 to 7, nothing is mentioned about the kinetics of the compound when it is administered to patients.
In designing pharmaceutical compositions containing a peptide or a protein, it is important to ensure the stability of the peptide or the protein prior to administration since peptides and proteins are generally unstable in aqueous solutions. It is, however, more important to design a preparation that can effectively elicit the activity of the administered compound.
Patent Document 1: International Patent Publication No. WO 01/07475
Patent Document 2: International Patent Publication No. WO 03/097083
Patent Document 3: Published Japanese translation of PCT application No. 2004-514651
Patent Document 4: Japanese Patent Publication No. Sho 63-40166
Patent Document 5: Japanese Patent No. 2643426
Patent Document 6: Published Japanese translation of PCT application No. 2004-522803
Patent Document 7: Japanese Patent Publication No. Hei 2-19092
Patent Document 8: Japanese Patent Publication No. 5-24129
Patent Document 9: Japanese Patent No. 3120987
Non-Patent Document 1: Kojima at al., Nature, vol. 402, pp. 656-660, 1999
Non-Patent Document 2: Tokihiro et al., J. Pharm. Pharmacol., vol. 52, pp. 911-917, 2000